Abstract: Ophiobolin A is sesterterpenoid-type phytotoxin with potential for weeds and diseases control. The restriction enzyme-mediated integration (REMI) was used to introduce the plasmid pSH75 into the ophiobolin A-producing Helminthosporium gramineum. Mutants with lower toxicity were selected with bioassay of the cell-free cultural filtrates. And the amount of ophiobolin A of lower toxicity mutants was detected with HPLC. In comparison with wild-type strain, the cell-free cultural filtrates of 6 REMI transformants showed significantly less inhibition on mycelial growth of plant pathogen Rhizoctoni solani, and therefore were categorized into lower toxicity mutants. Among the 6 strains, strains B014 and B016 produced no detectable ophiobolin A as measured with HPLC. Strains H002 and B018 produced less ophiobolin A in comparison with wild-type strain. These mutants might be useful for tagging and cloning genes related to ophiobolin A production in the future. This research also suggested REMI as a potential approach for improving the production of ophiobolin A by H.gramineum via genetic engineering.